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bioRxiv · 10.1101/2025.02.21.639412

Economical Method to Construct a Prism-based TIRF Setup on an Existing Confocal Microscope to Perform smFRET Experiments

Agarwala, P.·Sasmal, D. K.
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Abstract

Total internal reflection fluorescence (TIRF) microscopy enables observation of complex bio-assemblies and macromolecular dynamics in high spatial-temporal resolution at single-molecule level in real-time. With TIRF illumination, fluorophores near a sample substrate are excited by an evanescent field, thereby circumventing the axial diffraction limit of light. Prism-based TIRF (p-TIRF) microscopes are comparatively easy to use. They may be readily adjusted to meet the requirements of a broad range of experimental applications, such as to examine of macromolecular complexes, to study of the behaviour of vesicles and small organelles, the study of protein-DNA complexes at the single-molecule level. These experiments can give unique insights into the mechanisms driving the molecular interactions that underline many fundamental activities within the cell by providing information on fluctuation distributions and unusual events. Here, we report a detailed method to build a p-TIRF setup inexpensively using an existing confocal microscope where the same light source can be used for both systems. Furthermore, we provide a brief overview that aims to give the readers a stepwise tutorial protocol for building, assembling, aligning, and preparing the specimen to conduct single-molecule fluorescence resonance energy transfer (smFRET) experiments using a custom-built p-TIRF setup. We believe that this article will be of assistance to labs that already have a confocal microscope and want to perform TIRF experiments for potential future applications. Research HighlightWe report here a simple and cost-effective method to build a prism-based TIRF setup on a laser scanning confocal microscope. The setup is affordable, and users can use both confocal and TIRF modes in the same setup with the same light source and optical components. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/639412v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@e476e5org.highwire.dtl.DTLVardef@139d7b8org.highwire.dtl.DTLVardef@81ac21org.highwire.dtl.DTLVardef@14bd8f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Agarwala, P., Sasmal, D. K.. 2025-02-24. Economical Method to Construct a Prism-based TIRF Setup on an Existing Confocal Microscope to Perform smFRET Experiments. https://doi.org/10.1101/2025.02.21.639412

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